Infectious bursal disease virus VP2 fusion protein expressed by baculovirus

ABSTRACT

The present invention relates to the expression of the variable region of a VP2 protein from infectious bursal virus disease by recombinant baculovirus, diagnostic assays and vaccines containing the same.

This application is a continuation of application Ser. No. 08/148,252,filed on Nov. 4, 1993, now abandoned.

TECHNICAL FIELD

The present invention relates to the poultry industry, and inparticular, poultry viruses and vaccines therefrom.

The present invention relates to recombinant DNA-directed synthesis ofcertain proteins and the cell lines which express the recombinant DNAand proteins. More particularly, the present invention relates to theexpression of the variable region of a truncated VP2 protein frominfectious bursal disease virus by a recombinant baculovirus.

The present invention is useful for diagnosing infectious bursal diseasein poultry and as an infectious bursal disease vaccine containing thevariable region of the truncated VP2 protein.

BACKGROUND OF THE INVENTION

Infectious bursal disease, also known as Gumboro disease, infectspoultry world-wide and is responsible for many losses in the poultryindustry. Infectious bursal disease is known to be caused by a virus,the infectious bursal disease virus (IBDV). This disease often affectspoultry as young as one to six weeks old and causes inflammation,diarrhea, muscular hemorrhaging, bleeding, damage to the immune systemand necrosis of the Bursa of Fabricii.

The immunosuppression results from a depletion of the chicken's Blymphocytes (Burkhardt E., Muller H. (1987) Susceptibility of chickenblood lymphocytes and monocytes to infectious bursal disease virus(IBDV). Arch Virol 94:297-303). Secondary infections are commonlyassociated with infectious bursal disease (Cervantes H. M., Munger L.L., Ley D. H., Ficken M. D. (1988) Staphylococcus-induced gangrenousdermatitis in broilers. Avian Dis 32:140-142; Hofacre C. L., French J.D., Fletcher O. J. (1986) Subcutaneous clostridial infection inbroilers. Avian Dis 30:620-622; Rosenberger J. K., Gelb Jr. J (1977)Response to several avian respiratory viruses as affected by infectiousbursal disease virus. Avian Dis 22:95-105; Rosenberger J. K., Klopp S.,Eckroade R. J., Krauss W. C. (1975) The role of the infectious bursalagent and several avian adenoviruses in the hemorrhagic-aplastic-anemiasyndrome and gangrenous dermatitis. Avian Dis 19:717-729; Wyeth P. J.(1975) Effect of infectious bursal disease on the response of chickensto s typhimurium and E coli infections. Vet Rec 96:238-243). The IBDVvirion consists of two segments of double-stranded RNA (Muller H.,Scholtissek C., Becht H. (1979) The genome of infectious bursal diseasevirus consists of two segments of double-stranded RNA. J Virol31:584-589). One segment, called segment A, encodes a fusion proteinwhich is cleaved into the structural proteins VP2, VP3 and VP4 (HudsonP. J., McKern N. M., Power B. E., Azad A. A. (1986) Genomic structure ofthe large RNA segment of infectious bursal disease virus. Nucleic AcidRes 14:5001-5012). The other segment, segment B, encodes the putativeviral polymerase VP1 (Morgan M. M., Macreadie I. G., Harley V. R.,Hudson P. J., Azad A. A. (1988) Sequence of the small double-strandedRNA genomic segment of infectious bursal disease virus and its deduced90-kDa product. Virol 240-242). A neutralizing monoclonal antibody (MAb)binding site was mapped to a region of VP2 between amino acids 206 and350 (Azad A. A., Jagadish M. N., Fahey K. J. (1987) Deletion mapping andexpression in Escherichia coli of the large genomic segment of abirnavirus. Virol 161:145-152). Analysis of the nucleic acid andpredicted amino acid sequences among several strains of IBDV hasrevealed an area of high variability among strains of IBDV within thisregion (amino acids 239-332) (Bayliss C. D., Spies K., Peters R. W.,Papageorgiou A., Muller H., Boursnell M. E. G. (1990) A comparison ofthe sequences of segment A of four infectious bursal disease virusstrains and identification of a variable region in VP2. J Gen Virol71:1303-1312).

Several expression systems have been utilized to produce the IBVD VP2protein. Azad et al. (Azad A., Macreadie I., Vaughan P., Jagadish M.,McKern N., Heine H. G., Failla P., Ward C. (1990) Full protectionagainst an immunodepressive viral disease by a recombinant antigenproduced in yeast. Vacc 90:59-62) expressed VP2 as a fused and non-fusedprotein in Escherichia coil. Both expression products were poorimmunogens. A recombinant fowlpox virus expressing VP2 protected birdschallenged with IBDV against mortality, but not against damage to thebursa (Bayliss C. D., Peters R. W., Cook J. K. A., Reece R. L., HowesK., Binns M. M., Boursnell M. E. G. (1991) A recombinant fowlpox virusthat expresses the VP2 antigen of infectious bursal disease virusinduces protection against mortality caused by the virus. Arch Virol120:193-205). A recombinant VP2 protein produced in yeast inducedneutralizing antibodies and protected progeny chickens from IBDVchallenge (Hofacre et al., 1986, supra). IBDV antigen could not bedetected in the bursa of progeny from birds immunized with therecombinant yeast protein following challenge with the virus.Recombinant proteins expressed in baculovirus have been produced inamounts of 1 to 500 mg/ml (Luckow V. A., Summers M. D. (1988) Trends inthe development of baculovirus expression vectors. Biotech 6:47-56;Smith G. E., Summers M. D., Fraser M. J. (1983) Production of human betainterferon in insect cells infected with a baculovirus expressionvector. Mal Cell Biol 3:2156-2165). This expression system utilizes manyof the protein modification, processing and transport systems that occurin higher eukaryotic species.

Immunologic studies involving IBDV have suggested that VP2 contains aconformational dependent neutralizing epitope which could be used todistinguish serotypes (Becht, H., Muller H. K. (1988) Comparativestudies on structural and antigenic properties of two serotypes ofinfectious bursal disease virus. J Gen Virol 69:631-40). A monoclonalantibody (1/A6) against VP2 has been mapped to a small highly variableregion (Azad et al., 1987, supra).

IBDV proteins have also been expressed using baculovirus (Vakharia V.N., Snyder D. B., He J., Edwards G. H., Savage P. K., Mengel-Whereat S.A. (1993) Infectious bursal disease virus structural proteins expressedin a baculovirus recombinant confer protection in chickens. J Gen Virol74:1201-1206), wherein it was reported that these proteins reacted withIBDV specific monoclonal antibodies and resembled the native viralproteins. Seventy-nine percent of the birds vaccinated using thesebaculovirus expressed VP2, VP3 and VP4 proteins were protected against ahomologous IBDV challenge.

In addition, other vaccines have been produced in an attempt to controlinfectious bursal disease. These vaccines, developed containing eitherlive or attenuated viruses, or cultures of the bursal cells for example,while protecting the poultry from mortality, still cause symptoms ofinfectious bursal disease in the vaccinated poultry. These symptomsincluded growth retardation and injury to the Bursa of Fabricii. Anotherproblem with the prior art vaccines is that there are increasingoutbreaks of infectious bursal disease which are not prevented fromoccurrence by any of the prior art vaccines, Another problem is theimmunization itself of the poultry. Both the timing and method ofadministration of the vaccine continue to present problems. Inparticular, problems arise with attempts to vaccinate newly hatchedpoultry since such newly hatched poultry are often hatched with maternalantibodies to the infectious bursal disease (either to previousinfections or vaccination of the hen against the infectious bursaldisease). Thus, the time period for vaccination becomes critical. It isimportant to effectively and rapidly vaccinate large numbers of poultry.The mass immunization of poultry is best effected through such methodsof spraying or in the drinking water of the poultry.

Therefore, there are still problems in the prior art to determine thecauses of new outbreaks of infectious bursal disease and to develop avaccine which is resistant to both the known strains and further strainsof infectious bursal disease. There is a further need to determine amethod of preventing such further outbreaks through vaccination of theaffected population. There is a further need to develop a vaccine whichcan be administered in a timely manner, both by achieving vaccination atthe critical times in the poultry's life and by mass immunization torapidly inoculate the entire poultry population. There is still afurther need to provide a vaccine which does not cause symptoms ofinfectious bursal disease to occur in the vaccinated poultry.

DISCLOSURE OF INVENTION

According to the present invention, a portion of VP2 which contains thehighly variable region was isolated and expressed in a baculovirussystem. Further, according to the present invention, there is disclosedcells transfected with a DNA sequence in coding a VP2 fusion protein andcapable of expressing said protein.

The production of IBDV antigen for diagnostic assays from whole viralparticles is a costly endeavor. The VP2 fusion protein expressed by arecombinant baculovirus of the present invention is useful as animproved diagnostic reagent in, for example, ELISA, agar gel precipitinassay or other assays which detect IBDV antibodies in chickens. Thisfusion protein is also useful for further evaluating the neutralizingepitopes of the VP2 protein.

In particular, a portion of the VP2 gene from the variant A strain ofinfectious bursal disease virus was ligated into the pAc360 transfervector and transfected into baculovirus. Recombinant baculoviruses wereidentified using dot blot hybridization. According to one aspect of thepresent invention, one recombinant baculovirus, 9A5, expressed a 56.7kDa fusion protein. Radioimmunoprecipitation was used to confirm theidentity of this protein. Polyclonal antibodies against the MD strain ofIBDV immunoprecipitated the ³⁵ S-methionine labeled fusion protein. Thefusion protein was expressed from suspension cultures of Sf9 insectcells in quantities of at least about and often greater than 100mg/literof cell culture media. Specific-pathogen-free white leghorn chickenswere inoculated twice with 5×10⁶ or 1×10⁷ Sf9 insect cells that wereinfected with recombinant baculovirus expressing the VP2 fusion protein.Neutralizing antibody titers were observed in these birds following thesecond inoculation using a virus neutralization assay.

The present invention, thus, relates to novel cell lines such asrecombinant baculovirus 9A5, which express a VP2 fusion protein. Thesenovel cells line substantially enhance the availability of VP2 fusionprotein.

One object of the present invention is to provide purified forms of theVP2 fusion protein. An additional object of the present invention is toprovide purified forms of the VP2 fusion protein and novel cell linesexpressing the VP2 fusion protein, both which are valuable in theproduction of antigens for diagnostic assays. The VP2 fusion protein andthe cell lines expressing this protein are also valuable in theproduction of a new and improved vaccine against infectious bursaldisease.

Other objects, advantages and novel features of the present inventionwill be presented in the following detailed description and examples ofthe present invention.

DESCRIPTION OF THE INVENTION

The present invention has made it possible to express a VP2 structuralprotein in baculovirus. Until the present invention, no one has produceda fusion protein of a VP2 protein and baculovirus polyhedrosis protein.

The recombinant baculovirus 9A5 of the present invention expresses a56.7 kDa fusion protein. The VP2 gene fragment is 944 bases in lengthand does not include the initial 300 bases which results in the absenceof 100 amino acids located at the amino terminus of native VP2. Theabsence of these amino acids does not interfere with the proper foldingof the variable region.

The elicitation of neutralizing antibodies according to the presentinvention shows that proper folding of the VP2 fusion protein occurred.However, the titers of these neutralizing antibodies were lower in mostbirds and absent in others. As a result of this protein being a fusionprotein, 14 amino acids at the amino terminus and 184 amino acids at thecarboxy terminus flanked the 314 amino acids of the VP2 protein. Thetotal addition of 198 irrelevant amino acids may have affected theconformational dependent epitope and resulted in the observed lowneutralizing titers. However, it is believed that there are additionalconformationally dependent neutralizing epitopes Oppling V., Muller H.,Becht H. (1991) Heterogeneity of the antigenic site responsible for theinduction of neutralizing antibodies in infectious bursal disease virus.Arch Virol 119:211-223; Crisman J. M., Jackwood R. J., Lana D. P.,Jackwood D. J. (1992) Evaluation of VP2 epitopes of infectious bursaldisease virus using in vitro expression and radioimmunoprecipitation.Arch Virol 128:333-344). The inventors herein believe that it ispossible that other neutralizing epitopes are required to obtain highneutralizing antibody titers. Vakharia et al., supra, used baculovirusto express the entire VP2 protein in addition to the VP3 and VP4proteins of IBDV and obtained an average virus neutralizing antibodytiter of 1024 in chickens. The inventors thus believe that more thanjust the neutralizing epitope on VP2 described by Azad et al., supra, isneeded to elicit adequate immunity to IBDV. No one until the presentinventors herein has developed a recombinant baculovirus expressing thevariable region of the VP2 gene from the variant A strain of infectiousbursal disease.

The principles for the present invention will be explained by thisdetailed description of the preferred embodiment together with thefollowing examples.

Identification of the VP2 fusion protein

The 944 bp Pst I/Bal I fragment represents bases 434 through 1377 of theVP 2 gene which contains the variable sequence region (Bayliss et al,1990, supra). The recombinant transfer vector, p360v17, was identifiedusing restriction enzyme analysis and transfected into baculovirus DNA.Recombinant baculovirus plaques that were not refractive whenilluminated by a fiber optics light source (Dolan-Jenner Industries,Inc., Woburn, Mass.) were selected. Dot blot hybridization confirmed thepresence of the 944 bp fragment in recombinant viruses following eachplaque purification.

To determine if the recombinant viruses were expressing the VP2 fusionprotein, viral proteins were metabolically labeled in Sf9 cells at 48hours post inoculation (PI) with ³⁵ S-methionine and analyzed by 12%SDS-PAGE (O'Reilly D. R., Miller L. K., Luckow V. A. (1992) Baculovirusexpression vectors: a laboratory manual. W. H. Freeman and Company, NewYork). A protein with the size of the VP2 fusion protein (56.7 kDa) wasobserved in recombinant baculovirus 6A3 and 9A5. A similar size proteinwas not observed in the wild type baculovirus. Furthermore, thepolyhedrosis protein (32 kDa) was observed in the wildtype virus, butnot in the recombinant viruses.

Polyclonal chicken serum against the variant IBDV strain, MD, was usedto verify the authenticity of the 56.7 kDa protein in aradioimmunoprecipitation assay. Immunoprecipitated proteins wereseparated by 12.5 % SDS-PAGE and visualized using autoradiography. A56.7 kDa protein band was observed. This size band corresponded to thatof the fusion protein. No proteins were observed in the lanes containingprotein samples from the wildtype baculovirus and mock-infected cellcontrols.

The quantity of VP2 fusion protein in Coomassie blue stained gels wasdetermined by densitometer analysis and direct comparison to known BSAquantities. Expressed recombinant protein was produced in quantities>100mg/liter of insect media. Sf9 cells inoculated with recombinant 9A5 at aMOI of 0.02 and harvested 72 hours later produced 30 ug of the VP2fusion protein in 1×10⁷ cells. Sf9 cells inoculated with recombinant 9A5at a MOI of 0.2 and harvested at 72 hours PI produced>600 ug of the VP2fusion protein in 1×10⁷ cells. The intensity of recombinant 9A5 proteinbands stained with Coomassie blue was greater in samples harvested at 72hours than at 48 hours.

Immunogenic properties of the VP2 fusion protein

The ability of the VP2 fusion protein to elicit neutralizing antibodieswas determined by immunizing specific pathogen-free white leghornchickens with Sf9 cells infected with recombinant 9A5 baculovirus. Serafrom these birds were analyzed using a virus neutralization assay.

Titers of virus neutralizing antibodies were variable. Neutralizingantibodies were first detected at three weeks following the initialinoculation. Two weeks after the booster inoculation, the followingneutralizing antibody titers were determined in assays utilizing thecell culture adapted variant A strain of IBDV as antigen: two birds hadtiters of 320, one bird each had titers of 40, 20, 14 and 10. Four birdsinoculated with recombinant 9A5 baculovirus infected Sf9 cells had nodetectable antibodies at two weeks after the booster inoculation.Neutralizing antibody titers were similar for assays utilizing cellculture adapted variant IBDV strains IN or MD as antigen. All controlbird serum samples were negative for neutralizing antibodies.

Use of VP2 fusion protein useful in the production of antigens fordiagnostic assays

The VP2 fusion protein expressed by the recombinant baculovirus 9A5 isan improved diagnostic reagent for such assays as ELISA, agar gel,precipitin assays and other assays which detect IBDV antibodies inchickens. The VP2 fusion protein is also useful for evaluating theneutralizing epitopes of the VP2 protein.

According to one aspect of the present invention, a method forillustrating neutralizing antibodies to infectious bursal disease viruscomprises the steps of inoculating an animal susceptible to infectiousbursal disease with an effective amount of at least one of thefollowing: 1) a recombinant baculovirus expressing the VP2 fusionprotein or 2) the VP2 protein itself.

The concentration of VP2 protein in infected poultry differs fromnon-infected poultry. The reagent of this invention can be in the formof an absorbent resin having absorbed on a surface thereof an amount ofthe anti-VP2 fusion protein effective to render the resin reactive.Also, other non-reactive, preferably particulate resins which absorbproteins on an exterior surface thereof, such as those used toimmobilize enzymes can be used. The amount of antigen absorbed thereoncan range from about 5 to 100 nm/mg of resins and preferably about 25 to75 nm/mg.

Thus, the present invention also provides for a kit for detecting thepresence of a neutralizing antibody to infectious bursal virus disease.The kit preferably comprises a predetermined amount of an infectiousdisease virus antigen. It is to be understood that the antigen wouldpreferably be labelled with a readily determinable label to aid in thedetection of the infectious bursal disease virus. It is also understoodthat the kit can comprise a container for ease in mixing the sample fromthe tested poultry with the antigen.

The present invention contemplates the development of diagnostic assaysfor the determination of the neutralizing IBDV antibody. Those skilledin the art recognize that methods by which such assays are developed andutilized can be employed in the present invention and do not constitutean inventive aspect of the present invention. It is further understoodthat those skilled in the art will be able to utilize one of the variousmethods available for labeling antigens in order to perform diagnosticassays, such as competitive ELISA. Various labels or tags includingmaterials such as radioactive iodine and radioactive cobalt, biotin,various enzymes and fluorescing materials. It is understood that thelabeled antigen may be supported or unsupported and that suchdetermination can be best made by the practitioner. Further, thestandard for any labeled antigen is readily determined either directlyor indirectly. In a direct determination, the amount of antibody presentin the sample to the labeled antigen is determined directly bydetermining the amount of the bound material isolating and determiningthat amount of bound material. An indirect determination evaluation ismade of the amount of unbound materials left after a predetermined time.These determinations are compared with a previously established standardsuch that the presence of the neutralizing antibodies in the affectedpoultry are measured.

As will be recognized by those skilled in the art after establishing theneutralizing and binding capacity of the antigens, it is seen that theantigens can be used as a basis for diagnostic assays and testing forthe presence of infectious bursal disease in poultry populations.Further, the VP2 fusion can be used to inoculate poultry populationsagainst IBDV infection. It is known to those skilled in the art that themethod of inoculation can vary greatly. Such considerations involve theuse of either live or inactivated vaccine.

The vaccine may be prepared according to common methods, which are notper se a part of this invention. These methods involve the use of eitherlive or attenuated vaccines. These methods include heat and chemicalkilling which allow the vaccinated poultry to develop protectiveantibodies itself without it coming to the disease. It is also possibleto attenuate the viruses using such known methods as serial passage or acloning of the virus leading sequences of nucleic acids, orsite-directed mutagenesis. These methods provide a live non-virulentvaccine. It is understood that those of ordinary skill in the art willbe able to determine the actual amount of protection needed andtherefore, the level of protection needed by a particular poultrypopulation. The appropriate dosage levels can be readily determinedthrough routine experimentation to deliver the appropriate concentrationlevels to each poultry population. Further, it is understood that thevaccine may be prepared by incorporation of the virus derivative in aneffective carrier either by suspension or mixture or other knownmethods. The necessary levels of protection can range between onemicrogram to one milligram. Alternatively, vaccinations carried out overtime can be utilized having smaller effective dosages administered tothe poultry population. As is understood, the concentration dosagelevels needed to effectively inoculate poultry depends on the age andincrease in size of each poultry. It is understood that the vaccinecomprises the active ingredient and pharmaceutically acceptable carriersor diluents. In preferred embodiments, the carrier or diluents arecompatible with the vaccine administration procedures used with massvaccine administration procedures, such as spraying the animal or theanimal's environment or in the animal's drinking water. It iscontemplated, however, that the other administration methods such asinjections, eye drops (ophthalmically), nose drops (nasally) and thelike are also useful with the present invention. In addition, variousadjuvants are useful in order to enhance the poultry's immune responseto the antigens. These adjuvants are well known to those skilled in theart. Further, stabilizers and other ingredients are contemplated asbeing useful in preparing an acceptable vaccine for immunization.

The following examples give particular embodiments of the invention anddemonstrate the practice and advantages of the present invention. It isunderstood that the examples are given by way of illustration and arenot intended to limit the specification or claims in any manner.

EXAMPLES Viruses and antisera

Wildtype baculovirus (Autographa califonica nuclear polyhedrosis virus)was propagated in Spodoptera frugiperda (Sf9) cells (Invitrogen, SanDiego, Calif.). Cell culture-adapted variant IBDV strains IN, MD and A(Ismail N. M., Saif Y. M., Wigle W. L., Havenstein G. B., Jackson C.(1990)Infectious bursal disease virus variant from commercial leghornpullets. Avian Dis .34:141-145; Saif Y. M., Jackwood M. W., Jackwood D.H. (1987) Relatedness of the IBDV vaccine strains and field strains.Proc 36th Western Poultry Disease Conference, University of CaliforniaCooperative Extension, Davis Calif. pp.100-111); Rosenberger J. K.,Cloud S. S., Metz A. (1987) Use of infectious bursal disease virusvariant vaccines in broiler and broiler breeders. Proc Western PoultryDisease Conference, Davis, Calif. pp. 105-106) were propagated on babyGrivet monkey kidney (BGM-70) cells (Jackwood D. H., Saif Y. M., HughesJ. H. (1986) Replication of infectious bursal disease virus incontinuous cell lines. Avian Dis 31:370-375). Antisera against variantIBDV strain MD was collected from convalescent specific-pathogen-freewhite leghorn chickens.

Insertion of the VP2 gene into pAc360 transfer vector

The VP2 cDNA was provided by D. P. Lana (National Cancer InstituteFrederick Cancer Research and Development Center, Frederick, Md.). TheV17 cDNA was 1006 base pairs (bp) and contained bases 380 to 1386 of theVP2 gene (Lana D. P., Beisel C. E., Silva R. F. (1992) Geneticmechanisms of antigenie variation in infectious bursal disease virus:analysis of a naturally occurring variant virus. Virus Genes6-3:247-259). This fragment was ligated into pGem 3Zf(+) (PromegaCorporation, Madison, Wis.) (Crisman, et al., supra). The resultingrecombinant plasmid (pV-17) was transformed into Escherichia coil andplated on Luria agar plates containing 50 ug/ml ampicillin (SigmaChemical Co., St. Louis, Mo.). An isolated colony was selected and grownin terrific broth (Sambrook J., Fritsch E. F., Maniatis T. (1989)Molecular cloning: 2 laboratory manual, 2nd edn. Cold Spring HarborLaboratory Press, New York) containing 50 ug/ml ampicillin. Plasmid DNAwas isolated using an alkaline extraction procedure (Sambrook et al.,supra).

Plasmid pV-17 was digested with Ball (Promega) and partially digestedwith PstI (Promega). The DNA fragments were separated by electrophoresisusing 1% agarose (FMC Corporation, Rockland, Me.)in TBE buffer (89 mMTris base [pH 8.0], 89 mM boric acid, 2 mM EDTA) (Amresco, Solon, Ohio).A 944 bp fragment was cut from the gel and isolated into TBE bufferusing an electro-eluter (Bio-Rad Laboratories, Richmond, Calif.)according to the manufacturer's instructions. The 944 bp fragment wasextracted with phenol and chloroform, precipitated using ethanol andafter centrifugation was suspended in double distilled H₂ O. Theprotruding 3' Pst I end was removed using T4 DNA polymerase (Stratagene,LaJolla, Calif.) (Sambrook et al., supra). A BamH I 10mer linker wasligated onto each end using T4 Ligase (Bethesda Research Laboratories,Gaithersburg, Md.) as described in Sambrook et al., supra, The fragmentswere then digested to completion with BamH I (Promega).

Removal of linker fragments and isolation of the 954 bp fragment wasperformed by electrophoresis using 1.25 % SeaPlaque low gellingtemperature agarose (FMC Corporation) in TAE (40 mM Tris-acetate [ph8.0], 1 mM EDTA) (Amresco). The resulting 954 bp fragment was excisedfrom the gel and ligated into the transfer vector using an in-gelligation procedure (Ausubel F. M., Brent B., Kingston R. E., Moore D.D., Seidman J. G., Smith J. A., Struhl K. (1992) Short protocols inmolecular biology, 2nd edn. Greene Publishing Associates and John Wiley& Sons, New York).

Prior to ligation, the pAc360 Vector was digested with BamH I (Promega)and treated with shrimp alkaline phosphatase (United States BiochemicalCorporation, Cleveland, Ohio). The treated pAc360 DNA was extracted withphenol and chloroform, precipitated using ethanol and resuspended in TE(10 mM Tris-HCl [pH 7.6], I mM EDTA). Ligation reaction products weretransformed into E. coil Max Efficiency DH5α™ competent cells (BethesdaResearch Laboratories) according to the manufacturer's instructions. E.coli were plated on Luria agar plates containing 50 ug/ml ampicillin(Sigma). Selected colonies were grown in terrific broth with 50 ug/mlampicillin. Plasmid DNA from these colonies was isolated using amini-prep plasmid extraction procedure (Sambrook et al., supra).Restriction enzymes BamH I, Hind III, Pst I and Sal I were used toconfirm the orientation of the VP2 fragment.

Transfection and selection of recombinant virus

Transfection was conducted as described (MAXBAC™ baculovirus expressionsystem, Invitrogen). Recombinant plaques were identified visually andprepared for dot blot hybridization (Summers M. D., Smith G. E. (1988) Amanual of methods for baculovirus vectors and insect cell cultureprocedures. Texas Agricultural Experimental Station bulletin no. 1555.Texas Agricultural Experimental Station, College Station, Tex.). A 612bp Pst I/Bal I fragment within the 944 bp fragment was radiolabeledusing ³² P-dCTP (ICN Radiochemicals, Irvine, Calif.) andnick-translation (Bethesda Research Laboratories). Dot blothybridization was conducted (Jackwood D. J., Kibenge F., Mercado C.,(1989) Detection of infectious bursal disease virus by using cloned cDNAprobes. J Clin Microbiol 27:2437-2443). Positive baculoviruses whichcontained the VP2 gene fragment were plaque purified an additional twotimes. Following each plaque purification, hybridization was used toconfirm the presence of the VP2 gene fragment. Recombinant baculoviruseswere propagated on Sf9 cells for 48 hours. Viral proteins werevisualized using 12% SDS-PAGE and Coomassie blue staining (O'Reilly etal., supra). These proteins were also metabolically labeled with35S-methionine and analyzed using 12% SDS-PAGE.

The recombinant baculovirus designated 9A5 was propagated at amultiplicity of infection of 0.02 or 0.2 in Sf9 cells for 72 hours.Cells were propagated in suspension cultures as described in O'Reilly etal., supra. At 72 hours post infection, the Sf9 cells were counted todetermine cell density and then homogenized and stored at -70° C.Following 12% SDS-PAGE, quantities of the VP2 fusion protein weredetermined using a densitometer (Bio Rad, Richmond, Calif.). Sincebovine serum albumin (BSA) was similar in size to the VP2 fusionprotein, known quantities of BSA were separated by electrophoresis andused to prepare a standard curve. BSA samples were used at the followingquantities: 20 ug, 10 ug, 5 ug, 2.5 ug and 1 ug. Linear regressionanalysis of the absorption readings was used to determine the quantityof VP 2 fusion protein.

Radioimmunoprecipitation

VP2 fusion protein was ³⁵ S-methionine labeled (O'Reilly et al., supra).Cells infected with 9A5 were lysed using a double-detergent lysis bufferwith protease inhibitors (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.02%sodium azide, 0.1% SDS, 100 ug/ml phenylmethylsulfonyl, 1 ug/mlpepstatin, 0.5 ug/ml leupeptin, 1% Nonidet P-40). A 5 ul volume oflysate was mixed with 5 ul polyclonal MD antiserum. Incubation was at 4°C. for 3 hours. Following incubation, the mixture was combined with 2 ulof polyclonal rabbit anti-chicken serum (Sigma Chemical Company) andincubated at 4° C. for 1 hour. This mixture was added to 100 ul of a 1:1(v/v) mixture of Sepharose A and NET buffer (50 mM Tris-HCL, [pH 8.0];0.14M NaCl; 5 mM EDTA; 0.05% (v/v) Nonidet P-40) and shaken gently for1.25 hours at 4° C. The mixture was washed three times with NET bufferand suspended in SDS-loading buffer. Samples were analyzed using 12.5 %SDS-PAGE. Gels were vacuum dried and then exposed to X-Omat AR film(Eastman Kodak Company, Rochester, N.Y.).

Immunogenicity of baculovirus expressed VP2

Six-week-old specific-pathogen-free white leghorn chickens, were used todetermine the immunogenicity of the expressed VP2 fusion protein. Tenbirds were inoculated subcutaneously with 1×10⁷ recombinant 9A5baculovirus-infected Sf9 cells emulsified in Freund's complete adjuvant(Difco Laboratories, Detroit, Mich.). Nine control birds were inoculatedsubcutaneously with PBS emulsified in Freund's complete adjuvant (DifcoLaboratories). Birds were injected again two weeks later with thecorresponding inoculum emulsified in Freund's incomplete adjuvant (DifcoLaboratories). Blood samples were taken at 0, 14, 21, and 28 days afterthe initial injection. Sera from these samples were analyzed by aconstant-virus varying-antibody neutralization assay conducted in BGM-70cells as described (Jackwood D. H., Saif T. M. (1987) Antigenicdiversity of infectious bursal disease virus. Avian Dis 31:766-770)using the IN or MD IBDV strains as antigen.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications, as would be obvious to one having ordinary skill in thearts, may be made without departing from the scope of the invention,which is set forth in the claims appended hereto.

We claim:
 1. A fusion protein comprising a 314 amino acid sequenceencoded by the PstI-BalI fragment of the VP2 gene of IBDV variant A,said PstI-BalI fragment containing bases 434-1377 of the VP2 gene, andsaid fusion protein further comprising sequences from the baculoviruspolyhedrin protein encoded by a PAC360 vector.
 2. The fusion protein ofthe claim 1 wherein the fusion protein has 14 amino acids of thepolyhedrin sequence on the amino terminus of the VP2 sequence, and 184amino acids of the polyhedrin sequence on the carboxy terminus of theVP2 sequence.
 3. A baculovirus expressing the fusion protein of claim 1or 2.